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1.  Autovaccination Confers Protection against Devriesea agamarum Associated Septicemia but Not Dermatitis in Bearded Dragons (Pogona vitticeps) 
PLoS ONE  2014;9(12):e113084.
Devrieseasis caused by Devriesea agamarum is a highly prevalent disease in captive desert lizards, resulting in severe dermatitis and in some cases mass mortality. In this study, we assessed the contribution of autovaccination to devrieseasis control by evaluating the capacity of 5 different formalin-inactivated D. agamarum vaccines to induce a humoral immune response in bearded dragons (Pogona vitticeps). Each vaccine contained one of the following adjuvants: CpG, incomplete Freund's, Ribi, aluminium hydroxide, or curdlan. Lizards were administrated one of the vaccines through subcutaneous injection and booster vaccination was given 3 weeks after primo-vaccination. An indirect ELISA was developed and used to monitor lizard serological responses. Localized adverse effects following subcutaneous immunization were observed in all but the Ribi adjuvanted vaccine group. Following homologous experimental challenge, the incomplete Freund's as well as the Ribi vaccine were observed to confer protection in bearded dragons against the development of D. agamarum associated septicemia but not against dermatitis. Subsequently, two-dimensional gelelectrophoresis followed by immunoblotting and mass spectrometry was conducted with serum obtained from 3 lizards that showed seroconversion after immunisation with the Ribi vaccine. Fructose-bisphosphate aldolase and aldo-keto reductase of D. agamarum reacted with serum from the latter lizards. Based on the demonstrated seroconversion and partial protection against D. agamarum associated disease following the use of formalin-inactivated vaccines as well as the identification of target antigens in Ribi vaccinated bearded dragons, this study provides promising information towards the development of a vaccination strategy to control devrieseasis in captive lizard collections.
PMCID: PMC4257540  PMID: 25479609
2.  Genome-wide Association Study of Obsessive-Compulsive Disorder 
Stewart, S Evelyn | Yu, Dongmei | Scharf, Jeremiah M | Neale, Benjamin M | Fagerness, Jesen A | Mathews, Carol A | Arnold, Paul D | Evans, Patrick D | Gamazon, Eric R | Osiecki, Lisa | McGrath, Lauren | Haddad, Stephen | Crane, Jacquelyn | Hezel, Dianne | Illman, Cornelia | Mayerfeld, Catherine | Konkashbaev, Anuar | Liu, Chunyu | Pluzhnikov, Anna | Tikhomirov, Anna | Edlund, Christopher K | Rauch, Scott L | Moessner, Rainald | Falkai, Peter | Maier, Wolfgang | Ruhrmann, Stephan | Grabe, Hans-Jörgen | Lennertz, Leonard | Wagner, Michael | Bellodi, Laura | Cavallini, Maria Cristina | Richter, Margaret A | Cook, Edwin H | Kennedy, James L | Rosenberg, David | Stein, Dan J | Hemmings, Sian MJ | Lochner, Christine | Azzam, Amin | Chavira, Denise A | Fournier, Eduardo | Garrido, Helena | Sheppard, Brooke | Umaña, Paul | Murphy, Dennis L | Wendland, Jens R | Veenstra-VanderWeele, Jeremy | Denys, Damiaan | Blom, Rianne | Deforce, Dieter | Van Nieuwerburgh, Filip | Westenberg, Herman GM | Walitza, Susanne | Egberts, Karin | Renner, Tobias | Miguel, Euripedes Constantino | Cappi, Carolina | Hounie, Ana G | Conceição do Rosário, Maria | Sampaio, Aline S | Vallada, Homero | Nicolini, Humberto | Lanzagorta, Nuria | Camarena, Beatriz | Delorme, Richard | Leboyer, Marion | Pato, Carlos N | Pato, Michele T | Voyiaziakis, Emanuel | Heutink, Peter | Cath, Danielle C | Posthuma, Danielle | Smit, Jan H | Samuels, Jack | Bienvenu, O Joseph | Cullen, Bernadette | Fyer, Abby J | Grados, Marco A | Greenberg, Benjamin D | McCracken, James T | Riddle, Mark A | Wang, Ying | Coric, Vladimir | Leckman, James F | Bloch, Michael | Pittenger, Christopher | Eapen, Valsamma | Black, Donald W | Ophoff, Roel A | Strengman, Eric | Cusi, Daniele | Turiel, Maurizio | Frau, Francesca | Macciardi, Fabio | Gibbs, J Raphael | Cookson, Mark R | Singleton, Andrew | Hardy, John | Crenshaw, Andrew T | Parkin, Melissa A | Mirel, Daniel B | Conti, David V | Purcell, Shaun | Nestadt, Gerald | Hanna, Gregory L | Jenike, Michael A | Knowles, James A | Cox, Nancy | Pauls, David L
Molecular psychiatry  2012;18(7):788-798.
Obsessive-compulsive disorder (OCD) is a common, debilitating neuropsychiatric illness with complex genetic etiology. The International OCD Foundation Genetics Collaborative (IOCDF-GC) is a multi-national collaboration established to discover the genetic variation predisposing to OCD. A set of individuals affected with DSM-IV OCD, a subset of their parents, and unselected controls, were genotyped with several different Illumina SNP microarrays. After extensive data cleaning, 1,465 cases, 5,557 ancestry-matched controls and 400 complete trios remained, with a common set of 469,410 autosomal and 9,657 X-chromosome SNPs. Ancestry-stratified case-control association analyses were conducted for three genetically-defined subpopulations and combined in two meta-analyses, with and without the trio-based analysis. In the case-control analysis, the lowest two p-values were located within DLGAP1 (p=2.49×10-6 and p=3.44×10-6), a member of the neuronal postsynaptic density complex. In the trio analysis, rs6131295, near BTBD3, exceeded the genome-wide significance threshold with a p-value=3.84 × 10-8. However, when trios were meta-analyzed with the combined case-control samples, the p-value for this variant was 3.62×10-5, losing genome-wide significance. Although no SNPs were identified to be associated with OCD at a genome-wide significant level in the combined trio-case-control sample, a significant enrichment of methylation-QTLs (p<0.001) and frontal lobe eQTLs (p=0.001) was observed within the top-ranked SNPs (p<0.01) from the trio-case-control analysis, suggesting these top signals may have a broad role in gene expression in the brain, and possibly in the etiology of OCD.
PMCID: PMC4218751  PMID: 22889921
Obsessive-compulsive disorder; GWAS; Genetic; Genomic; Neurodevelopmental disorder; DLGAP
3.  Illumina sequencing of 15 deafness genes using fragmented amplicons 
BMC Research Notes  2014;7(1):509.
Resequencing of deafness related genes using GS FLX massive parallel sequencing of PCR amplicons spanning selected genes has previously been reported as a successful strategy to discover causal variants. The amplicon lengths were designed to be smaller than the sequencing read length of GS FLX technology, but are longer than Illumina sequencing technology read lengths. Fragmentation is thus required to sequence these amplicons using high throughput Illumina technology.
We performed Illumina sequencing in 4 patients on 563 multiplexed amplicons covering the exons of 15 genes involved in the hearing process. After exploring several fragmentation strategies, the amplicons were fragmented using Covaris sonication prior to library preparation. CLC genomic workbench was used to analyze the data.
We achieve an excellent coverage with more than 99% of the amplicons bases covered. All variants that were previously validated using Sanger sequencing, were also called in this study. Variant calling revealed less false positive and false negative results compared to the previous study. For each patient, several variants were found that are reported by ClinVar as possible hearing loss variants.
Migration from GS FLX amplicon sequencing to Illumina amplicon sequencing is straightforward and leads to more accurate results.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-0500-7-509) contains supplementary material, which is available to authorized users.
PMCID: PMC4266979  PMID: 25106482
4.  Quantitative Proteomics to Characterize Specific Histone H2A Proteolysis in Chronic Lymphocytic Leukemia and the Myeloid THP-1 Cell Line 
Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation.
PMCID: PMC4100102  PMID: 24871368
histone H2A; proteolysis; histone clipping; chronic lymphocytic leukemia; THP-1 cells; quantitative proteomics
5.  Draft Genome Sequence of Methicillin-Resistant Staphylococcus epidermidis Strain ET-024, Isolated from an Endotracheal Tube Biofilm of a Mechanically Ventilated Patient 
Genome Announcements  2014;2(3):e00527-14.
Staphylococcus epidermidis strain ET-024 was isolated from a biofilm on an endotracheal tube of a mechanically ventilated patient. This strain is resistant to methicillin, and the draft genome sequence shares some characteristics with other nosocomial S. epidermidis strains (such as S. epidermidis RP62A).
PMCID: PMC4038896  PMID: 24874691
6.  Presence and Analysis of Plasmids in Human and Animal Associated Arcobacter Species 
PLoS ONE  2014;9(1):e85487.
In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.
PMCID: PMC3896396  PMID: 24465575
7.  Perfrin, a novel bacteriocin associated with netB positive Clostridium perfringens strains from broilers with necrotic enteritis 
Veterinary Research  2014;45(1):40.
Necrotic enteritis in broiler chickens is associated with netB positive Clostridium perfringens type A strains. It is known that C. perfringens strains isolated from outbreaks of necrotic enteritis are more capable of secreting factors inhibiting growth of other C. perfringens strains than strains isolated from the gut of healthy chickens. This characteristic could lead to extensive and selective presence of a strain that contains the genetic make-up enabling to secrete toxins that cause gut lesions. This report describes the discovery, purification, characterization and recombinant expression of a novel bacteriocin, referred to as perfrin, produced by a necrotic enteritis-associated netB-positive C. perfringens strain. Perfrin is a 11.5 kDa C-terminal fragment of a 22.9 kDa protein and showed no sequence homology to any currently known bacteriocin. The 11.5 kDa fragment can be cloned into Escherichia coli, and expression yielded an active peptide. PCR detection of the gene showed its presence in 10 netB-positive C. perfringens strains of broiler origin, and not in other C. perfringens strains tested (isolated from broilers, cattle, sheep, pigs, and humans). Perfrin and NetB are not located on the same genetic element since NetB is plasmid-encoded and perfrin is not. The bacteriocin has bactericidal activity over a wide pH-range but is thermolabile and sensitive to proteolytic digestion (trypsin, proteinase K). C. perfringens bacteriocins, such as perfrin, can be considered as an additional factor involved in the pathogenesis of necrotic enteritis in broilers.
PMCID: PMC3992141  PMID: 24708344
8.  Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua 
BMC Plant Biology  2013;13:220.
The medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level.
By laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).
A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells.
Our results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.
PMCID: PMC3878173  PMID: 24359620
Artemisia annua; Artemisinin; RNASeq; Glandular trichomes; Filamentous trichomes; Laser microdissection pressure catapulting; MEP pathway; Mevalonate pathway; Lipid biosynthesis; Terpene biosynthesis
9.  Partitioning the Heritability of Tourette Syndrome and Obsessive Compulsive Disorder Reveals Differences in Genetic Architecture 
Davis, Lea K. | Yu, Dongmei | Keenan, Clare L. | Gamazon, Eric R. | Konkashbaev, Anuar I. | Derks, Eske M. | Neale, Benjamin M. | Yang, Jian | Lee, S. Hong | Evans, Patrick | Barr, Cathy L. | Bellodi, Laura | Benarroch, Fortu | Berrio, Gabriel Bedoya | Bienvenu, Oscar J. | Bloch, Michael H. | Blom, Rianne M. | Bruun, Ruth D. | Budman, Cathy L. | Camarena, Beatriz | Campbell, Desmond | Cappi, Carolina | Cardona Silgado, Julio C. | Cath, Danielle C. | Cavallini, Maria C. | Chavira, Denise A. | Chouinard, Sylvain | Conti, David V. | Cook, Edwin H. | Coric, Vladimir | Cullen, Bernadette A. | Deforce, Dieter | Delorme, Richard | Dion, Yves | Edlund, Christopher K. | Egberts, Karin | Falkai, Peter | Fernandez, Thomas V. | Gallagher, Patience J. | Garrido, Helena | Geller, Daniel | Girard, Simon L. | Grabe, Hans J. | Grados, Marco A. | Greenberg, Benjamin D. | Gross-Tsur, Varda | Haddad, Stephen | Heiman, Gary A. | Hemmings, Sian M. J. | Hounie, Ana G. | Illmann, Cornelia | Jankovic, Joseph | Jenike, Michael A. | Kennedy, James L. | King, Robert A. | Kremeyer, Barbara | Kurlan, Roger | Lanzagorta, Nuria | Leboyer, Marion | Leckman, James F. | Lennertz, Leonhard | Liu, Chunyu | Lochner, Christine | Lowe, Thomas L. | Macciardi, Fabio | McCracken, James T. | McGrath, Lauren M. | Mesa Restrepo, Sandra C. | Moessner, Rainald | Morgan, Jubel | Muller, Heike | Murphy, Dennis L. | Naarden, Allan L. | Ochoa, William Cornejo | Ophoff, Roel A. | Osiecki, Lisa | Pakstis, Andrew J. | Pato, Michele T. | Pato, Carlos N. | Piacentini, John | Pittenger, Christopher | Pollak, Yehuda | Rauch, Scott L. | Renner, Tobias J. | Reus, Victor I. | Richter, Margaret A. | Riddle, Mark A. | Robertson, Mary M. | Romero, Roxana | Rosàrio, Maria C. | Rosenberg, David | Rouleau, Guy A. | Ruhrmann, Stephan | Ruiz-Linares, Andres | Sampaio, Aline S. | Samuels, Jack | Sandor, Paul | Sheppard, Brooke | Singer, Harvey S. | Smit, Jan H. | Stein, Dan J. | Strengman, E. | Tischfield, Jay A. | Valencia Duarte, Ana V. | Vallada, Homero | Van Nieuwerburgh, Filip | Veenstra-VanderWeele, Jeremy | Walitza, Susanne | Wang, Ying | Wendland, Jens R. | Westenberg, Herman G. M. | Shugart, Yin Yao | Miguel, Euripedes C. | McMahon, William | Wagner, Michael | Nicolini, Humberto | Posthuma, Danielle | Hanna, Gregory L. | Heutink, Peter | Denys, Damiaan | Arnold, Paul D. | Oostra, Ben A. | Nestadt, Gerald | Freimer, Nelson B. | Pauls, David L. | Wray, Naomi R. | Stewart, S. Evelyn | Mathews, Carol A. | Knowles, James A. | Cox, Nancy J. | Scharf, Jeremiah M.
PLoS Genetics  2013;9(10):e1003864.
The direct estimation of heritability from genome-wide common variant data as implemented in the program Genome-wide Complex Trait Analysis (GCTA) has provided a means to quantify heritability attributable to all interrogated variants. We have quantified the variance in liability to disease explained by all SNPs for two phenotypically-related neurobehavioral disorders, obsessive-compulsive disorder (OCD) and Tourette Syndrome (TS), using GCTA. Our analysis yielded a heritability point estimate of 0.58 (se = 0.09, p = 5.64e-12) for TS, and 0.37 (se = 0.07, p = 1.5e-07) for OCD. In addition, we conducted multiple genomic partitioning analyses to identify genomic elements that concentrate this heritability. We examined genomic architectures of TS and OCD by chromosome, MAF bin, and functional annotations. In addition, we assessed heritability for early onset and adult onset OCD. Among other notable results, we found that SNPs with a minor allele frequency of less than 5% accounted for 21% of the TS heritability and 0% of the OCD heritability. Additionally, we identified a significant contribution to TS and OCD heritability by variants significantly associated with gene expression in two regions of the brain (parietal cortex and cerebellum) for which we had available expression quantitative trait loci (eQTLs). Finally we analyzed the genetic correlation between TS and OCD, revealing a genetic correlation of 0.41 (se = 0.15, p = 0.002). These results are very close to previous heritability estimates for TS and OCD based on twin and family studies, suggesting that very little, if any, heritability is truly missing (i.e., unassayed) from TS and OCD GWAS studies of common variation. The results also indicate that there is some genetic overlap between these two phenotypically-related neuropsychiatric disorders, but suggest that the two disorders have distinct genetic architectures.
Author Summary
Family and twin studies have shown that genetic risk factors are important in the development of Tourette Syndrome (TS) and obsessive compulsive disorder (OCD). However, efforts to identify the individual genetic risk factors involved in these two neuropsychiatric disorders have been largely unsuccessful. One possible explanation for this is that many genetic variations scattered throughout the genome each contribute a small amount to the overall risk. For TS and OCD, the genetic architecture (characterized by the number, frequency, and distribution of genetic risk factors) is presently unknown. This study examined the genetic architecture of TS and OCD in a variety of ways. We found that rare genetic changes account for more genetic risk in TS than in OCD; certain chromosomes contribute to OCD risk more than others; and variants that influence the level of genes expressed in two regions of the brain can account for a significant amount of risk for both TS and OCD. Results from this study might help in determining where, and what kind of variants are individual risk factors for TS and OCD and where they might be located in the human genome.
PMCID: PMC3812053  PMID: 24204291
10.  The Prevalence of Nine Genetic Disorders in a Dog Population from Belgium, the Netherlands and Germany 
PLoS ONE  2013;8(9):e74811.
The objective of this study was to screen a dog population from Belgium, the Netherlands and Germany for the presence of mutant alleles associated with hip dysplasia (HD), degenerative myelopathy (DM), exercise-induced collapse (EIC), neuronal ceroid lipofuscinosis 4A (NCL), centronuclear myopathy (HMLR), mucopolysaccharidosis VII (MPS VII), myotonia congenita (MG), gangliosidosis (GM1) and muscular dystrophy (Duchenne type) (GRMD). Blood samples (K3EDTA) were collected for genotyping with Kompetitive Allele Specific PCR (n = 476). Allele and genotype frequencies were calculated in those breeds with at least 12 samples (n = 8). Hardy-Weinberg equilibrium was tested. Genetic variation was identified for 4 out of 9 disorders: mutant alleles were found in 49, 15, 3 and 2 breeds for HD, DM, EIC and NCL respectively. Additionally, mutant alleles were identified in crossbreeds for both HD and EIC. For HD, DM, EIC and NCL mutant alleles were newly discovered in 43, 13, 2 and 1 breed(s), respectively. In 9, 2 and 1 breed(s) for DM, EIC and NCL respectively, the mutant allele was detected, but the respective disorder has not been reported in those breeds. For 5 disorders (HMLR, MPS VII, MG, GM1, GRMD), the mutant allele could not be identified in our population. For the other 4 disorders (HD, DM, EIC, NCL), prevalence of associated mutant alleles seems strongly breed dependent. Surprisingly, mutant alleles were found in many breeds where the disorder has not been reported to date.
PMCID: PMC3777979  PMID: 24069350
11.  Ancient Dispersal of the Human Fungal Pathogen Cryptococcus gattii from the Amazon Rainforest 
PLoS ONE  2013;8(8):e71148.
Over the past two decades, several fungal outbreaks have occurred, including the high-profile ‘Vancouver Island’ and ‘Pacific Northwest’ outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.
PMCID: PMC3737135  PMID: 23940707
12.  Full Genome Sequences of All Nine Chlamydia psittaci Genotype Reference Strains 
Journal of Bacteriology  2012;194(24):6930-6931.
Chlamydia psittaci primarily infects birds, but zoonotic transmission occurs in people in close contact with infected birds. The clinical outcome ranges from inapparent disease to pneumonia. Here we report the genome sequences of all 9 Chlamydia psittaci genotype reference strains.
PMCID: PMC3510619  PMID: 23209198
13.  Genome Sequence of Helicobacter heilmannii Sensu Stricto ASB1 Isolated from the Gastric Mucosa of a Kitten with Severe Gastritis 
Genome Announcements  2013;1(1):e00033-12.
Here we report the genome sequence of Helicobacter heilmannii sensu stricto ASB1 isolated from the gastric mucosa of a kitten with severe gastritis. Helicobacter heilmannii sensu stricto has also been associated with gastric disease in humans. Availability of this genome sequence will contribute to the identification of genes involved in the pathogen’s virulence and carcinogenic properties.
PMCID: PMC3569310  PMID: 23405321
14.  Assessment of Microbial Diversity in Biofilms Recovered from Endotracheal Tubes Using Culture Dependent and Independent Approaches 
PLoS ONE  2012;7(6):e38401.
Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.
PMCID: PMC3367921  PMID: 22693635
15.  Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform 
BMC Medical Genomics  2012;5:17.
Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.
In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.
We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.
PMCID: PMC3443074  PMID: 22607986
Deafness; Next generation sequencing; PCR based enrichment; Genetic diagnostics
16.  Fetal Microchimeric Cells in Blood of Women with an Autoimmune Thyroid Disease 
PLoS ONE  2011;6(12):e29646.
Hashimoto's thyroiditis (HT) and Graves' disease (GD), two autoimmune thyroid diseases (AITD), occur more frequently in women than in men and show an increased incidence in the years following parturition. Persisting fetal cells could play a role in the development of these diseases.
Aim of this study was to detect and characterize fetal cells in blood of postpartum women with and without an AITD.
Eleven patients with an AITD and ten healthy volunteers, all given birth to a son maximum 5 years before analysis, and three women who never had been pregnant, were included. None of them had any other disease of the thyroid which could interfere with the results obtained.
Fluorescence in situ hybridization (FISH) and repeated FISH were used to count the number of male fetal cells. Furthermore, the fetal cells were further characterized.
In patients with HT, 7 to 11 fetal cells per 1.000.000 maternal cells were detected, compared to 14 to 29 fetal cells in patients with GD (p = 0,0061). In patients with HT, mainly fetal CD8+ T cells were found, while in patients with GD, fetal B and CD4+ T cells were detected. In healthy volunteers with son, 0 to 5 fetal cells were observed, which was significantly less than the number observed in patients (p<0,05). In women who never had been pregnant, no male cells were detected.
This study shows a clear association between fetal microchimeric cells and autoimmune thyroid diseases.
PMCID: PMC3246474  PMID: 22216337
17.  Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination 
Nucleic Acids Research  2011;40(3):e24.
Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3–5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome.
PMCID: PMC3273786  PMID: 22127871
18.  Quantitative Bias in Illumina TruSeq and a Novel Post Amplification Barcoding Strategy for Multiplexed DNA and Small RNA Deep Sequencing 
PLoS ONE  2011;6(10):e26969.
Here we demonstrate a method for unbiased multiplexed deep sequencing of RNA and DNA libraries using a novel, efficient and adaptable barcoding strategy called Post Amplification Ligation-Mediated (PALM). PALM barcoding is performed as the very last step of library preparation, eliminating a potential barcode-induced bias and allowing the flexibility to synthesize as many barcodes as needed. We sequenced PALM barcoded micro RNA (miRNA) and DNA reference samples and evaluated the quantitative barcode-induced bias in comparison to the same reference samples prepared using the Illumina TruSeq barcoding strategy. The Illumina TruSeq small RNA strategy introduces the barcode during the PCR step using differentially barcoded primers, while the TruSeq DNA strategy introduces the barcode before the PCR step by ligation of differentially barcoded adaptors. Results show virtually no bias between the differentially barcoded miRNA and DNA samples, both for the PALM and the TruSeq sample preparation methods. We also multiplexed miRNA reference samples using a pre-PCR barcode ligation. This barcoding strategy results in significant bias.
PMCID: PMC3203936  PMID: 22046424
19.  Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics 
PLoS ONE  2011;6(9):e25531.
Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics.
PMCID: PMC3184136  PMID: 21980484
20.  Complete Nucleotide Sequence of CTX-M-15-Plasmids from Clinical Escherichia coli Isolates: Insertional Events of Transposons and Insertion Sequences 
PLoS ONE  2010;5(6):e11202.
CTX-M-producing Escherichia coli strains are regarded as major global pathogens.
Methodology/Principal Findings
The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp) from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp) from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: blaTEM-1 and blaCTX-M-15. It shares more than 90% homology with a previously published blaCTX-M-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, blaTEM-1 and blaCTX-M-15, were found. Six resistance genes, blaTEM-1, blaCTX-M-15, blaOXA-1, aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the blaCTX-M-15-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a blaTEM-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the blaOXA-1, aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids.
Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of diverse multiresistant plasmids found in clinical Enterobacteriaceae.
PMCID: PMC2887853  PMID: 20585456
21.  Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline 
BMC Bioinformatics  2010;11:269.
Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions.
We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data.
We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.
PMCID: PMC2880033  PMID: 20487544
22.  Suspension fluorescence in situ hybridization (S-FISH) combined with automatic detection and laser microdissection for STR profiling of male cells in male/female mixtures 
Laser microdissection is a valuable tool for isolating specific cells from mixtures, such as male cells in a mixture with female cells, e.g., in cases of sexual assault. These cells can be stained with Y-chromosome-specific probes. We developed an automatic screening method to detect male cells after fluorescence in situ hybridization in suspension (S-FISH). To simulate forensic casework, the method was tested on female saliva after cataglottis (a kiss involving tongue-to-tongue contact) and on licking traces (swabs of dried male saliva on female skin) even after drying. After isolation of the detected cells, short tandem repeat profiling was performed. Full DNA profiles could consistently be obtained from as little as ten buccal cells. Isolation of five cells resulted in a mean of 98% (SD of 3.4%) of the alleles detected, showing that the developed S-FISH staining had no significant negative influence on DNA recovery and can be used in forensic casework.
PMCID: PMC2754505  PMID: 19319556
Forensic science; Laser capture microdissection; Male non-sperm cells; Hybridization; Automatic cell recognition
23.  Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR 
Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts.
The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (β-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase β-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase).
Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours.
In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours.
PMCID: PMC1557526  PMID: 16889665
24.  Response to serotonin reuptake inhibitors in OCD is not influenced by common CYP2D6 polymorphisms 
The cornerstone of pharmacotherapy for OCD is serotonin reuptake inhibition, either with clomipramine or with selective serotonin reuptake inhibitors (SSRIs). In spite of the success of serotonin reuptake inhibiting drugs, nearly half of OCD patients do not respond to treatment. Treatment response may be affected by genetic polymorphisms of the P450 metabolic system. The four most common enzyme-activity reducing polymorphisms of the P450 CYP2D6 enzyme were determined in 91 outpatients with primary OCD according to DSM-IV criteria, receiving dosages titrated upward to 300 mg/day of venlafaxine or 60 mg/day of paroxetine, using a fixed dosing schedule. Our results show that the investigated CYP2D6 polymorphisms are not a decisive factor in the response to paroxetine and venlafaxine treatment in OCD in spite of their highly significant effect on the blood levels of these medicines.
PMCID: PMC2824234  PMID: 20174590
Obsessive-compulsive disorder; OCD; CYP2D6; venlafaxine; paroxetine; polymorphisms; response; blood levels; plasma levels

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